Shigella flexneri, a facultative intracellular pathogen, is a causative agent of shigellosis (bacterial dysentery) in humans. Basic research aimed at understanding the adaptation of this intracellular pathogen to the eukaryotic host cell will be useful for designing more effective therapies and will yield information applicable to other intracellular pathogens. Shigella interacts with several human cell types while within the human host including colonic epithelial cells and intestinal macrophage cells. The mechanisms by which the bacterium adapts to these environments are not completely understood, but require the ability to sense and respond accordingly to the current environment by altering bacterial gene expression. Thus, expression of a unique set of genes encoding proteins that help the bacterium survive and/or multiply within the eukaryotic cell is induced when Shigella is intracellular. Since Shigella virulence depends largely on the ability to evade the macrophage killing and multiply within the colonic epithelial cells, the long-term goal of the research program is to identify the physiological processes that Shigella employs to survive and/or multiply within these environments, as well as the elements that regulate Shigella gene expression in these environments. The goal of this particular project is to determine the contribution of six S. flexneri two component regulatory systems (TCRS) to Shigella adaptation to the eukaryotic cells and will be accomplished by the completion of three specific aims. The first undertaking (specific aim 1) will be to construct six mutant Shigella strains that lack particular TCRS (BaeS/BaeR, BarA/UvrY, EvgS/EvgA, NtrB/NtrC, CreC/CreB, and YfhK/YfhA) so that the contribution of these systems to Shigella virulence can be examined. Specific aim 2 is to analyze the effects of the deletion mutations in each TCRS on the ability of Shigella to survive and grow in epithelial cell culture by assessing plaque formation on cell monolayers. If a TCRS mutant does not form plaques or forms plaques that are smaller than those formed by the parental Shigella strain, this will suggest that the TCRS activates expression of a gene(s) that enhances the ability of Shigella to survive, multiply, or spread to adjacent epithelial cells. Specific aim 3 is to assess whether the six TCRS systems activate expression of a gene(s) that contribute to survival of Shigella within macrophage cells by analyzing each TCRS mutant for the ability to survive in macrophages and induce macrophage apoptosis. [unreadable] [unreadable] Shigella species are the causative agents of shigellosis (bacterial dysentery) in humans that results in 1.5 million cases in industrialized countries, 165 million cases in underdeveloped countries, and over one million deaths annually. Shigellosis is a frequent cause of diarrhea in US citizens traveling to underdeveloped countries and is of concern to US military operations, both because Shigella is a potential biowarfare agent and because shigellosis impacts troop health in countries where Shigella is endemic. The research described in this proposal will be useful for designing more effective therapies and will yield information applicable to other similar pathogens. [unreadable] [unreadable] [unreadable] [unreadable]